Crop Protection 77 (2015) 38e44

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Crop Protection

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Flea (Coleoptera: Chrysomelidae, Alticinae) in Bt- (MON810) and near isogenic maize stands: composition and activity densities in Hungarian fields * Agnes Szen asi a, , Viktor Marko b a Plant Protection Institute, Faculty of Agriculture and Environmental Sciences, Szent Istvan University 2100 God€ oll€ oP} ater K. str. 1, Hungary b Department of Entomology, Faculty of Horticultural Science, Corvinus University of Budapest 1118 Budapest Menesi str. 44, Hungary article info abstract

Article history: Flea beetles (Chrysomelidae, Alticinae) were collected with Pherocon AM yellow sticky traps in maize Received 2 April 2015 plots to compare the assemblages from transgenic Bt- (genetic event MON810, producing Cry1Ab protein Received in revised form effective against lepidopteran pests) and near isogenic maize in Hungary. Altogether, 51,348 flea 7 July 2015 individuals from 26 species were collected. The dominant species were atra (F.) and Phyllotreta Accepted 8 July 2015 vittula (Redtenbacher). Their abundances along with other (non-P. atra and non-P. vittula) flea beetle Available online 25 July 2015 species showed no significant differences between Bt- and isogenic maize plots. Similarly, no difference was found between Bt maize and isogenic maize plots in the species richness of the flea beetle Keywords: Flea beetles assemblages. © Bt maize 2015 Elsevier Ltd. All rights reserved. MON810 Species composition Phyllotreta atra Phyllotreta vittula

1. Introduction exposed to the Cry1Ab protein, e.g., the Cry1Ab toxin was detected in the flea beetle Chateocnema pulicaria Melsheimer feeding on The Cry1Ab toxin produced by maize hybrids containing MON810 maize (Harwood et al., 2005), although herbivores with MON810 event is highly effective in controlling target different types of mouthparts (chewing, sucking) may ingest larvae (Ostrinia nubilalis (Hübner), Pyralidae and Sesamia non- different amounts of Bt toxin. Also, predators consuming agrioides (Lefebvre), Noctuidae) and may also affect larvae of other phytophagous containing Cry1Ab toxin may move the toxin Lepidoptera species (e.g. Helicoverpa armigera (Hübner) [Eizaguirre into higher trophic levels (Harwood et al., 2005). et al., 2010; Kiss et al., 2003] Mythimna unipuncta (Haworth) European Union legislation requires a pre-market risk assess- [Eizaguirre et al., 2010; Pilcher et al., 1997]). However, maize fields ment of genetically modified crops before commercial use (EC, harbour species rich assemblages of other groups 2001, 2002; EFSA, 2010; EU, 2013). For that purpose, this study (Mesz aros et al., 1984), including flea beetles, which are present in examined potential effects of genetically modified maize express- maize stands in high numbers, occasionally causing damage in both ing the Cry1Ab toxin on non-target, within-maize herbivores, using Europe (Vor€ os€ and Maros, 2004) and North America (Hoffmann flea beetles as model species. et al., 1995). These phytophagous insects in maize may also serve Several studies have found no differences in abundance, sea- as prey for predacious species (Arpas et al., 2005). The presence of sonal activity and assemblage characteristics in several non-target the Cry1Ab protein in all maize plant parts throughout the whole herbivore taxa in Bt (MON810) and isogenic maize (Balog et al., growing season might affect a number of associated organisms 2010a,b; Bourguet et al., 2002; Daly and Buntin, 2005; Lozzia, besides the target pests. Herbivores feeding on maize may be 1999; Lozzia and Rigamonti, 1998; Musser and Shelton, 2003; Sehnal et al., 2004). These same authors have raised the impor- tance of longer term field studies to detect possible cumulative effects over several seasons, to determine thresholds for detecting * Corresponding author. E-mail address: [email protected] (A. Szen asi). any effects and for selecting suitable species for impact studies. The http://dx.doi.org/10.1016/j.cropro.2015.07.008 0261-2194/© 2015 Elsevier Ltd. All rights reserved. A. Szenasi, V. Marko / Crop Protection 77 (2015) 38e44 39 necessity of a more ecological approach in the study of the potential our study was to complete and supplement these data. impact of growing GM crops on non-target organisms is raised by Cry1Ab protein (MON810 event) shows high specificity to target Andow et al. (2006, 2013), Arpaia et al. (2014), Lovei€ and Arpaia organisms (Ostrinia nubilalis, Sesamia nonagrioides) under labora- (2005), Lovei€ et al. (2009). tory studies (Tier1), and variable mortality to larvae of numerous Several studies have been conducted in Hungary on economi- Lepidoptera species (MON810 SO Update, Perry et al., 2010, 2011) cally important maize pests, secondary pests, other herbivores, and (Tier1a and b, Tier2) and by maize hybrids on the field (Tier3) predators including a 10-year maize ecosystem research study (intended effect). However, only a few in-planta data (unintended (Mesz aros et al., 1984). This faunal survey, however, focused pri- effect) are available about flea beetles in connection with secondary marily on the occurrence of arthropod taxa in the studied maize metabolites and others (Bak et al., 1999; Nielsen et al., 2001; fields and did not collect data on their abundance. Because the Verpoorte and Memelink, 2002). The further purpose of this Cry1Ab toxin is expressed in all maize tissues sampling should study was to survey for possible un-intended effects of the MON810 examine all groups of herbivores in maize that occur at high enough maize on flea beetles. density to permit quantitative comparisons between Bt and isogenic maize. 2. Materials and methods Our study group, flea beetles are best known as pests of Bras- sicaceae crops (Bohinc et al., 2013; Saringer, 1990; Trdan et al., 2.1. Experimental site 2008). In contrast, Phyllotreta vittula (Redtenbacher) has been recorded as a pest on cereals (including maize) (Fritzsche, 1971; The two-year (2002 and 2003) field experiment was carried out Szeoke,} 1997), sugar beet and crucifers (Naibo, 1974), including in an isolated maize stand located near Budapest (GPS, N: 47 250; yellow mustard (Hurej et al., 1997). Vig (1998a) reported a wide E: 18 470), Hungary, surrounded by large stone fruit orchards host range of P. vittula including grasses, maize and Brassicaceae, (apricot, peach and plum). Plots within the experimental field were but damage in Hungary is reported only on cereals, such as maize arranged in a randomized complete block design with six replica- (Nagy and Deseo,} 1969; Szeoke,} 1997). Phyllotreta vittula has been tions. The plots (sized 30 30 m) were planted either with Bt maize found damaging maize in Hungary (Gyulai and Garay, 1996; (DK 440 BTY e transformation event MON810) or with its near Hinfner and Papp, 1961; Jablonowski, 1906; Nagy and Deseo,} isogenic line (DK 440) on chernozem soil. An alley distance of 3 m 1969; Szeoke} et al., 1996; Szeoke,} 1997; Szucs,} 1973; Vor€ os€ and was used between replications. A retention zone (a pollen capture Maros, 2004), in the former Soviet Union (Arnoldi and Gurjeva, crop surrounding the entire test field) was established to a maize 1960; Scsegolev, 1952), in Germany (Sorauer, 1954), in Poland hybrid of similar maturity ground to the test hybrid, in accordance (Kania, 1962), in the former Czechoslovakia (Hruska, 1962), in with the requirements of the release permit. France (Leclant, 1977), in the former Yugoslavia (Sekulic et al., 1989) Maize was planted at a seed rate of 65,000 seeds/ha and was and in Romania (Grozea et al., 2006), making it one of the most reduced to 50,000 plants/ha after emergence. Sowing was in late important Coleoptera species attacking maize foliage (Sekulic et al., April and maize was harvested in mid-October to early November 1989). -damage by P. vittula is most pregnant in spring on the depending on the year. juvenile plants (Saringer, 1990; Scsegolev, 1951; Szeoke} et al., 1996). In addition to P. vittula, aridula (Gyllenhal) is found 2.2. Sampling in Hungary on maize and other cereals (Saringer, 1990). The flea beetle (Harris) is an early season pest of maize in adults were collected with Pherocon AM yellow Kansas, USA, where chemical controls have occasionally been sticky traps (Trece Inc. California, USA), since yellow colour has long needed (Wilde et al., 2004). Chaetocnema pulicaria is a pest of maize been known as attractive for flea beetles (Hung and Hwang, 2000; in the USA (Steffey et al., 1999) and an important pest of sweet corn Vincent and Stewart, 1986). Three traps/plot were placed between in Iowa (Hoffmann et al., 1995). rows 20 and 21, 7.5 m apart and 7.5 m from the field edge. Traps In Hungary, dense flea beetle populations occur only in periods were fixed to a wooden stick at a height of maize canopy until of dry, sunny weather (Saringer, 1990), when young maize plants silking, when they were adjusted to ear height. In 2002, traps were are especially susceptible to injury if drought stressed (Szeoke} et al., changed weekly, and according to the experiences of the first year, 1996; Vor€ os€ and Maros, 2004). In older plants, flea beetles reduce in order to reduce efforts and costs of sampling, in 2003 they were foliar area and thus photosynthesis (Szeoke} et al., 1996). Under dry changed fortnightly. Sampling took place from late May to late conditions, damage increases and plant development is inhibited September in 2002 and from early June to mid-September in 2003. (Nagy and Deseo,} 1969). Among different crops the most conspic- Flea beetle adults were removed by petrol from the traps and uous injury by P. vittula is found in maize, mainly on the edges of submitted to I. Rozner (Museum of Natural History, Budapest) for fields and in unevenly emerged stands. In some cases, the lower identification. Collected adults were identified using the key of 1e4 of certain maize plants are totally destroyed (Gyulai and Warchalowsky (2003). Garay, 1996). Arthropod assemblages were surveyed in Bt- and in isogenic 2.3. Statistical analysis maize plots from 2001 to 2003 under an EU-5th framework project. In 2001, significant numbers of flea beetles and feeding damage The catches of all three yellow sticky traps per plot were pooled were observed on the maize plants in the study field. In the spring, for analysis. To compare the effect of different maize hybrids on flea adult flea beetles were feeding on the lower 1e5 leaves of maize beetle abundance and species richness, a repeated measures anal- plants, and caused notable damage in all three years of the study. ysis of variance (ANOVA) was performed. Yearly comparisons were This taxon was therefore selected for more detailed sampling with based on the samples collected at corresponding sampling dates ® Pherocon AM yellow sticky traps in 2002e2003, although only the (see ‘Maize hybrids’ versus ‘Sampling dates’); Welch's test was used species list of flea beetles collected in 2002 has already been pub- for the analysis of the main effect of ‘Maize hybrids’ and the lished (Kiss et al., 2003). GreenhouseeGeisser test (with epsilon-adjusted degrees of Since only a few studies (Grozea et al., 2006; Rauschen et al., freedom) was used for the trial factor (sampling dates) and for the 2010; Vrablova, 2002) have examined the species composition, ‘Maize hybrids’ trial interaction effect. The total number of Alti- dominance, and phenology of flea beetles in maize fields, the aim of cinae species collected during each year was compared with a one- 40 A. Szenasi, V. Marko / Crop Protection 77 (2015) 38e44 sample t test. A Welch test or (non-parametric) BrunnereMunzel Table 2 test was performed for comparison of abundances across sampling Mean number of Phyllotreta atra, P. vittula and other flea beetles (number of in- dividuals in a plot per sampling date, SE). Mean species richness (number of flea dates. All statistical analyses were performed with the software beetle species collected in a plot per sampling date, SE) and total species richness package ROPstat (Vargha et al., 2015). (total number of flea beetle species per plot in 2002 or 2003, SE). There was no difference between the maize hybrids in any comparison. For statistical analyses, see 3. Results Table 3. 2002 2003 Altogether, 51,348 flea beetle individuals from 26 species were Bt Iso Bt Iso captured by yellow sticky traps during 2002 and 2003 combined. In 2002, 18 flea beetle species were found in both Bt and isogenic P. atra 68.2 (6.2) 55.5 (5.7) 98.9 (10.6) 86.6 (6.7) P. vittula 57.4 (1.9) 53.4 (4.5) 315.3 (13.1) 313.1 (8.1) maize plots. In 2003, 17 species were found in Bt maize and 14 Other species 0.8 (0.1) 0.9 (0.1) 2.1 (0.2) 1.9 (0.2) species were found in isogenic maize (Tables 1 and 2). The highest Species rich./date 2.1 (0.1) 2.2 (0.1) 3.4 (0.1) 3.3 (0.1) species richness in 2002 was found from late June to mid-July, and, Total species rich. 8.3 (0.3) 8.2 (0.5) 9.3 (0.6) 8.5 (0.5) in 2003, from mid-June to late July. While species richness showed no difference between treatments, the number of captured species was significantly higher in Bt maize than in isogenic maize on 23 4. Discussion July 2002 (Welch t ¼ 2.739, df ¼ 5.0, p ¼ 0.041) and on 18 June 2003 (Welch t ¼ 2.272, df ¼ 9.1, p ¼ 0.049) (Fig. 1). In this study species-rich and abundant flea beetle assemblages The most abundant flea beetle species, in all Bt- and isogenic were found both in the Bt- (Cry1Ab) and in the isogenic maize plots. maize plots in both years, were Phyllotreta atra and P. vittula. Phyllotreta species were dominant in maize in both years. We found Together, these two species represented 99.3% and 99.5% of the P. vittula to be the most abundant species followed by P. atra, with total catch in 2002 and 2003 (Table 1). The highest trap catch of the other flea beetles species captured only in small numbers P. atra and P. vittula occurred between 18 June and 16 July (Table 1). Of the eight Alticinae species collected on maize by sweep depending on the year. In neither year was there a significant dif- netting in Slovakia by Vrablova (2002), all species except two ference in abundance of P. atra or of P. vittula between the Bt and (Chaetocnema laevicollis [Thomson] and Longitarsus pellucidus non-Bt maize varieties (Figs. 2 and 3). Neither was any difference [Foudras]) were found in our study as well. Phyllotreta vittula, observed for the other (non-P. atra and non-P. vittula) flea beetles Chaetocnema tibialis and P. atra were the dominant species in the (Fig. 4), although in 2002 the abundance of this group was signif- study in Slovakia, while P. vittula and P. atra were the dominant icantly higher in Bt maize on 16 July (Welch t ¼3.213, df ¼ 5.8, species in our study, in both Bt and non Bt maize. Phyllotreta vittula, p ¼ 0.019) and significantly higher in isogenic maize on the 23rd of P. atra and Chaetocnema aridula were also observed on maize in July (Welch t ¼ 2.739, df ¼ 5.0, p ¼ 0.041) (Fig. 4)(Table 3 and Romania (Grozea et al., 2006). Chaetocnema tibialis was also found Supplementary Table 1). on maize in both our study and in the USSR (Naibo, 1974). The maize is mentioned as food plant of C. aridula in Hungary (Saringer, 1990) which was detected in our field study as well, but only in low Table 1 number (Table 1). Species composition of flea beetle assemblages on Bt and isogenic maize (plots) and According to previous observations in Hungary (Saringer, 1990) the individual number of the species (Soskút, Hungary, 2002e2003, P. atra feeds on cultivated and wild growing cruciferous plants, on e June September). Reseda and Tropaeolum spp., on Cleome speciosissima and pea (Balas Species/year 2002 2003 and Saringer, 1982). In summer emerged flea beetle adults maintain Bt Iso Bt Iso their fecundity by feeding on cruciferous crops and weeds as well (Saringer, 1990). While no data is available on P. atra feeding on 1 Altica sp. 2 3 fi 2 Aphthona sp. 1 maize, high number of this species in the experimental maize eld 3 Chaetocnema aridula (Gyllenhal) 3 3 8 11 may be explained by the dispersion of the newly emerged adults 4 Chaetocnema concinna (Marsham) 5 3 9 4 from other crops. 5 Chaetocnema hortensis (Geoffroy) 2 1 10 10 In plant choice tests P. vittula preferred grasses (Agropyron 6 Chaetocnema tibialis (Illiger) 8 8 29 35 species), but sufficient feeding and egg laying was observed on 7 Epitrix pubescens (Koch) 2 3 8 Longitarsus exoletus (L.) 3 Brassicaceae and other Poaceae such as maize (Vig, 1998a). Earlier 9 Longitarsus melanocephalus (De Geer) 2 2 observations found that P. vittula disappeared from the maize fields 10 Longitarsus obliteratus (Rosenhauer) 1 from early-mid-July, but in our study it remained on the maize 11 Longitarsus parvulus (Paykull) 1 plants until the end of August in 2002 and until the middle of 12 Longitarsus pratensis (Pancer) 2 13 Longitarsus rubiginosus (Foudras) 3 6 1 September in 2003 (Fig. 3). However, P. atra reached its lowest 14 Longitarsus succineus (Foudras) 2 3 abundance much earlier, by the second decade of July and by the 15 Longitarsus sp. 1 1 7 1 mid-August in 2002 and 2003, respectively (Fig. 2). Flight activity 16 Phyllotreta aenea (Illiger) 1 1 peaks can be explained by the summer appearance of the newly Phyllotreta atra 17 (F.) 4909 3998 4153 3635 emerged adults (Foster, 1984; Hurej et al., 1997; Naibo, 1974; 18 Phyllotreta balcanica Heikertinger 3 19 Phyllotreta cruciferae (Goeze) 1 3 3 3 Saringer, 1990), as the summer generation adults of P. vittula and 20 Phyllotreta diademata Foudras 2 1 1 P. atra emerged at the end of June or beginning of July (Kocourek 21 (L.) 1 1 3 4 et al., 2002; Naibo, 1974). At this time the young P. vittula adults 22 Phyllotreta nigripes (F). 18 19 1 dispersed from cruciferous plants to cereal crops including maize 23 Phyllotreta procera (Redtenbacher) 1 3 (Leclant, 1977). However, Vig (1998b) found that the newly 24 Phyllotreta undulata Kutschera 3 7 6 25 Phyllotreta vittula (Redtenbacher) 4135 3842 13,243 13,150 emerged P. vittula adults appeared from late August to mid- 26 Psylliodes luteola (O.F. Müller) 1 September, significantly later than in other studies. Phyllotreta atra was the most abundant in the first decade of July No. of species 18 18 17 14 in both years, while P. vittula in mid-July in 2002, and from the mid- No. of individuals 9099 7905 17,482 16,862 June to early July in 2003 (Figs. 2 and 3). These periods overlap with A. Szenasi, V. Marko / Crop Protection 77 (2015) 38e44 41

Fig. 1. Mean species richness (number of species/3 traps/plot) of flea beetles collected by yellow sticky traps in Bt (BT) and isogenic (ISO) maize plots in 2002e2003, at Soskút, Hungary. Vertical lines indicate ± SE. Different letters within a date represents significant differences (p < 0.05).

Fig. 2. Mean activity density (number of individuals/3 traps/plot) of Phyllotreta atra, collected by yellow sticky traps in Bt (BT) and isogenic (ISO) maize plots in 2002e2003, at Soskút, Hungary. Vertical lines indicate ± SE. No differences between treatments on the same date were significant.

Fig. 3. Mean activity density (number of individuals/3 traps/plot) of Phyllotreata vittula, collected by yellow sticky traps in Bt (BT) and isogenic (ISO) maize plots in 2002e2003, at Soskút, Hungary. Vertical lines indicate ± SE. No differences between treatments on the same date were significant. 42 A. Szenasi, V. Marko / Crop Protection 77 (2015) 38e44

Fig. 4. Mean activity density (number of individuals/3 traps/plot) of Alticinae other than P. atra or P. vittula, collected by yellow sticky traps in Bt (BT) and isogenic (ISO) maize plots in 2002e2003, at Soskút, Hungary. Vertical lines indicate ± SE. Different letters within a date represents significant differences (p < 0.05).

Table 3 Statistical analyses (repeated measures ANOVA; Welch test for the effect of ‘maize hybrids’, GeissereGreenhouse test for the effect of ‘sampling dates’ and ‘maize hybrids’ ‘sampling date’ interaction) of the abundance of Phyllotreta atra, P. vittula, other (non-P. atra and non-P. vittula) flea beetle species and species richness of flea beetles per sampling date. One sample t test for the total species richness of flea beetles. See also Table 2.

Maize Epsilon Date Maize date

df F p df F p df F p

2002 P. atra 1; 9.9 1.130 0.3129 0.1 1.1; 11.0 87.118 0.0000 1.10; 10.96 1.038 0.3384 P. vittula 1; 6.7 0.351 0.5729 0.170 1.9; 18.7 65.888 0.0000 1.87; 18.72 0.270 0.7525 Other species 1; 10.0 0.663 0.4344 0.270 3.0; 29.7 7.862 0.0005 2.97; 29.65 2.067 0.1265 Spec. rich./date 1; 9.4 0.211 0.6565 0.404 4.0; 40.4 16.606 0.0000 4.04; 40.37 1.518 0.2148 Total spec. rich. 10 0.194a 0.8501 e e eee ee 2003 P. atra 1; 8.4 0.833 0.3868 0.355 2.1; 21.3 87.044 0.0000 2.13; 21.28 1.152 0.3376 P. vittula 1; 8.3 0.018 0.8970 0.323 1.9; 19.4 73.534 0.0000 1.94; 19.36 0.180 0.8305 Other species 1; 8.4 0.833 0.3868 0.355 2.1; 21.3 87.044 0.0000 2.13; 21.28 1.152 0.3376 Spec. rich./date 1; 9.4 0.265 0.6187 0.594 3.6; 35.7 13.268 0.0000 3.57; 35.65 1.927 0.1337 Total spec. rich. 10 1.000a 0.3409 e e eee ee

a t values (one sample t test). the first observed damage by P. vittula in different maize fields in no specific mechanism is known that would lead us to expect that Hungary, namely from the end of June to early July (Nagy and plant-incorporated Bt toxins would affect any non-target phytopha- Deseo,} 1969) and in Romania from mid-May to early July (Grozea gous without specific Bt protein binding sites, it is unclear et al., 2006). In 2003 the abundance of P. vittula was much higher whether the Bt effect on flea beetles in the study of Daly and Buntin than in 2002 (Table 1, Fig. 3) which might be due to the drought, (2005) was real or a statistical, or simply a sampling artefact. since a mass reproduction of the flea beetles can occur under such weather conditions (Saringer, 1990). Acknowledgements We found no adverse effect of Bt-maize (MON810 event) on flea beetle assemblages or species, or on their abundance or species This research was supported by EU-5th framework programme richness. In Germany, Chaetocnema spp., Longitarsus spp., Phyllotreta (Contract No: QLK3-CT-2000-00547) and by Research Centre of fl spp. and Psylliodes chrysocephala L. ea beetle species were collected Excellence-9878/2015/FEKUT (Szent Istvan University). The au- fi by sweep net and Phyllotreta spp. by panicle samples in a 3-year eld thors thank M. Perczel and Soskút Fruct Kft. for technical support study that compared MON88017 Bt with near isogenic maize. Similar and to I. Rozner (Hungarian Natural History Museum Budapest) for fl to our results, P. vittula was the most abundant ea beetle species and identifications. no significant difference was found between the Bt and non-Bt maize plots(Rauschen et al., 2010). In the same study, however, no flea beetle Appendix A. Supplementary data adults were observed with these sampling methods from MON810 and near isogenic maize (Nobilis) (Rauschen et al., 2010). Chae- Supplementary data related to this article can be found at http:// tocnema pulicaria wasamong the predominant herbivores in MON810 dx.doi.org/10.1016/j.cropro.2015.07.008. maize hybrid in Maryland, USA (Dively, 2005)andinGeorgia(Daly and Buntin, 2005). In the Maryland study, in agreement with our findings, flea beetles showed no difference between Bt and non-Bt References maize plots (Dively, 2005). In the Georgia study, the number of flea Andow, D.A., Lovei,€ G.L., Arpaia, S., 2006. Ecological risk assessment for Bt crops. beetles was higher in Bt than non-Bt maize, suggesting that they were Nat. Biotechnol. 24, 749e751. not adversely affected by the Bt maize (Daly and Buntin, 2005). While Andow, D.A., Lovei,€ G.L., Arpaia, S., Wilson, L., Fontes, E.M.G., Hilbeck, A., Lang, A., A. Szenasi, V. Marko / Crop Protection 77 (2015) 38e44 43

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